Malaria is a parasitic infection distributed widely in the subtropical zone. Its pathogen is a malaria parasite classified as apicomplexa and is transmitted by an anopheles mosquito.
Malaria is classified into 4 types, that is, tropical malaria, tertian malaria, quartan malaria and ovale malaria, among which tropical malaria is particularly malignant, and unless its therapy is initiated within a few hours after onset, tropical malaria is accompanied by severe symptoms and complications, resulting frequently in death. Other malaria, on the other hand, is not so severe and rarely leads to death.
Accordingly, tropical malaria and other malaria are considerably different in respect of treatment method and medicine to be used. Symptoms of tropical malaria become rapidly worse, thus making immediate treatment necessary, and from the viewpoint of saving a patient, there are cases where administration of medicine is initiated when the patient is suspected of having tropical malaria before the final diagnosis is not established. Accordingly, there is a possibility of wrong diagnosis and the side effect of the medicine administered. Other malaria does not require such immediate treatment and can thus be treated by spending much time.
In the stage of clinical diagnosis, therefore, it is very important to discriminate tropical malaria from other malaria rapidly and accurately thereby determining a suitable therapeutic method. Accordingly, early diagnosis of malaria, particularly tropical malaria, is important.
In treatment of tropical malaria, the type of medicine administered, the amount of the medicine administered and the administration span are empirically judged and determined by a physician on the basis of infection ratio (ratio of the number of malaria infected red blood cells to the number of total red blood cells contained in blood of a predetermined volume). In consideration of treatment of tropical malaria, it is therefore necessary to discriminate tropical malaria from other malaria, and it is also important to know the malaria infection ratio in blood from the patient.
As a conventional method used in detecting malaria, there is a method which comprises preparing a smear of blood collected from a predetermined object, subjecting it to Giemsa staining and observing it under a microscope to detect malaria infected red blood cells, or a method which comprises counting red blood cells and infected red blood cells in a specific visual field and calculating the infection ratio.
However, the method by observation under a microscope is troublesome because of necessity for the steps of preparing, fixing, staining and drying a smear. Further, sophisticated skills are necessary for discriminating malaria infected red blood cells from malaria uninfected red blood cells and for judging whether the type of malaria with which the cells were infected is tropical malaria or other malaria. In addition, much time (usually 15 minutes or more for one patient) is necessary for observation under a microscope.
As a method of automatically detecting malaria infected red blood cells, a method which involves staining malaria infected red blood cells with a fluorescent dye and detecting the malaria infected red blood cells with a flow cytometer is also developed.
However, when a nucleic acid fluorescent dye is used as described in the prior art referred to in U.S. Pat. No. 5,470,751, there is a problem that not only malaria infected red blood cells but also reticulocytes are stained, so the two kinds of cells cannot be discriminated from each other. Staining of only malaria infected red blood cells with a specific fluorescent dye (Auramine O analogue) at low concentration is proposed in U.S. Pat. No. 5,470,751 supra. In this method, however, discrimination of malaria uninfected red blood cells from infected red blood cells is not made sufficiently evident.
On the other hand, P. H. Vianen et al. (P. H. Vianen et al., Cytometry; 14:276-280) disclose a method which comprises lysing red blood cells with a lysing agent containing a buffer agent, formaldehyde and diethylene glycol, to release malaria parasites from the malaria infected red blood cells, staining the malaria parasites with a dye Hoechst 33258, and detecting the parasites with a flow cytometer. In this method, the influence of malaria uninfected red blood cells and reticulocytes is negligible, but there is a problem that the lysing and staining procedures are troublesome to require dozens of minutes or more.
JP-A 11-75892 discloses a method which comprises releasing malaria parasites by hemolysis, rapidly staining the malaria parasites specifically with a nucleic acid-staining dye and, without centrifugal separation, detecting them with a flow cytometer. However, discrimination between tropical malaria and other malaria is conducted generally by observing the form of malaria parasites in malaria infected red blood cells, so the method of JP-A 11-75892 supra wherein red blood cells are lysed can judge whether the cells are infected with malaria or not, but cannot determine whether the malaria is tropical malaria or not.
JP-A 2004-105027 proposes a method of judging the type of malaria parasite with a flow cytometer by utilizing a difference in the amount of a nucleic acid-binding dye between tropical malaria and other malaria. This method is a method which comprises lysing red blood cells in a sample to release malaria parasites, detecting the intensity of fluorescence from the malaria parasites in the measurement sample, and judging the type of the malaria parasites on the basis of the frequency distribution of malaria parasites having fluorescence intensity in a predetermined range.
However, the method of detecting malaria parasites released by lysing red blood cells, used in the above-mentioned references, that is, JP-A 11-75892, JP-A 2004-105027, and P. H. Vianen et al., Cytometry, 14:276-280, cannot determine accurate infection ratio for the following reason.
FIG. 1 shows a life cycle of malaria parasites in red blood cells, wherein malaria parasites which upon blood sucking by an anopheles mosquito, enter into the living body are released as merozoites from hepatocytes into blood, to enter into red blood cells thereby initiating the life cycle in red blood cells. In the life cycle in red blood cells, the malaria parasite grows into a ring form, trophozoite and schizont in this order, and the schizont when divided into a plurality of metozoites breaks the infected red blood cell to release merozoites from the red blood cell, and the merozoites released into blood enter other red blood cells and re-initiate the life cycle in red blood cells. The malaria parasite grows by repeating this cycle and continues breaking red blood cells in blood.
The red blood cell into which the ring form enters includes not only a red blood cell into which one ring formenters (referred to hereinafter as “ring form (single)”) but also a red blood cell into which two or more ring forms enter (referred to hereinafter as “ring form (multi)”). In the method of detecting parasites released by lysing red blood cells, however, a malaria parasite released from the ring form (single) and one of malaria parasites released from the ring form (multi) exhibit the same fluorescence intensity and thus cannot distinguished from each other. As a result, the ring form (multi) is regarded as a plurality of ring forms (single). There is also the case a schizont just before division may be regarded as being released from a plurality of red blood cells.
In the conventional method of observation under a microscope, on the other hand, the infection ratio is calculated assuming that the number of infected red blood cells is not affected by whether or not the ring forms (multi) exist and the schizont is in a matured stage just before division. Accordingly, the infection ratio obtained in the method which comprises lysing red blood cells and detecting released malaria parasites to determine the infection ratio on the basis of fluorescence intensity corresponding to the total amount of DNA in the malaria parasites does not agree with the infection ratio calculated in the conventional method of observation under a microscope. Particularly in the phenomenon in which a schizont just before division is divided by the influence of hemolysis into a plurality of metozoites or in tropical malaria where the proportion of a multi-infected red blood cell (that is, a red blood cell infected with 2 or 3 parasites) tends to be high, the infection ratio cannot be accurately grasped in the method directing attention to the frequency of released parasites obtained by lysing red blood cells. This means that in the medical field where a therapeutic method is selected and determined on the basis of the infection ratio calculated on the basis of the conventional method of observation under a microscope, tropical malaria can be judged, but selection of a suitable therapeutic method established up to now becomes difficult.